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1 Reprod Dev Biol 33(3) : (2009) , Changing Proteins in Granulosa Cells during Follicular Development in Pig In-Soon Chae 1, Dong-Min Jang 3, Hee-Tae Cheong 2, Boo-Keun Yang 1 and Choon-Keun Park 1, 1 College of Animal Life Sciences, 2 School of Veterinary Medicine, Kangwon National University, Chuncheon , Korea 3 CHA Stem Cell Institute, CHA Hospital, Seoul , Korea This study analyzed change of proteins in granulosa cells during the porcine follicuar development by proteomics techniques. Granulosa cells of the follicles, of which the diameter is 2 4 mm and 6 10 mm, were collected from ovary of slaughtered pig that each follicle of diameter 1 4 mm and 6 10 mm. We extracted glanulosa cell proteins by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 200 l. Immobilized ph gradient(ipg) strip used 18 cm, 3 10 NL. SDS-PAGE used 10% acrylamide gel. After silver staining, Melanie 7 and naked eye test were used for spot analyzation. Increasing proteins in glanulosa cell of 6 10 mm follicle were 7 spots. This spots were analyzed by MALDI-TOF MS and searched on NCBInr. In results, 7 spots were similar to zinc/ling finger protein 3 precursor (RING finger protein 203), angiomotin, heat shock 60 kda protein 1 (chaperonin) isoform 1 (HSP60), similar to transducin-like enhancer protein 1 (TLE 1), SH3 and PX domains 2A (SH3PXD2A). Those proteins were related with transfer between cells. Increase of proteins has an effect on follicular development. (Key words : Two-dimensional electrophoresis, MALDI-TOF, Granulosa cell, Protein profiling, Pig follicle) 포유동물난소의난포내에서난자의성숙과분화는체세포성난포세포와발달중인생식세포사이에긴밀하게관련되어있다 (Down, 1995). 난자와과립막세포사이의정보교환은상호지향적이며, 난자와난포의기능과발달을위해필수적이다 (Eppig, 1997). 이전의많은연구들에서난포발달의다양한측면들이난자의존재에의해영향을받는다는것이증명되었다 (Vanderhyden, 1996; Erickson 와 Shimasaki, 2001; Eppig 등, 2002; Matzuk 등, 2002). 난자특이적인성장인자인 BMP-15(bone morphogenetic protein-15) 는난포생성전기간에걸쳐랫 (Otsuka 등, 2000), 마우스 (Dube 등, 1998) 난자에서발현되는것으로보고되고있다. Otsuka 등 (2000) 은 BMK- 15 가체외에서 FSH- 독립성과립막세포분화의원인임을보여주었다. BMK-15 는또한성장분화인자 -9(GDF-9) 과밀접하게관련이있으며, 이러한두가지인자들은난소에유사한발현패턴을갖고있다 (Dube 등, 1998). 일반적으로 FSH 이외에난포막세포나과립막세포에의해분비된인접분비인자들이개별난포의발달을조절할수있기는하지만, 난자가난포발달의조절에지배적인역할을하는것으로생각된다 (Hsueh 등, 2000). 이외에줄기세포인자 (SCF; stem cell factor) 도난포발달에다양한역할을갖고있는것으로보고되고있다 (Yoshida 등, 1997). SCF 는난포보충과일차난포발달의초기진행에영향을미치며 (Parrott 와 Skinner, 1998), SCF mrna 는태아및성체난소에서의난포성숙의모든단계에서과립막세포에국한되어나타나는것으로알려져있다 (Tisdall 등, 1999). 이와같이포유동물의난포생성은체세포성난포세포또는성숙중인생식세포에의해분비되는다양한단백질인자들에의해조절되고있는것으로알려져있지만, 수많은연구에도불구하고난소기능조절에관여하는단백질과수용체에대한상관관계는완전히밝혀지지않았다. 따라서난포생성동안과립막세포의증식, 분화및성숙을조절하는메커니즘의이해를증진시키기위하여본연구에서는돼지에서난포생성에관여하는단백질인자 * 논문은 2007 년정부 ( 교육인적자원부 ) 의재원으로한국학술진흥재단의지원을받아수행된연구임 (KRF F00059). Corresponding author : Phone: , parkck@kangwon.ac.kr

184 채인순등 들을프로테오믹스기법을이용하여확인하고자하였다. 과립막세포단백질의수집도축직후의돼지에서난소를회수하여 37 의생리식염수에침적하여 2 3 시간이내에실험실로운반하였다. 난포액은 10 ml 주사기에 18G 주사침을이용하여직경 2 4 mm 의종속난포와 6 10 mm 의우세난포로부터각각흡입, 회수하였다.

2 184 채인순등 들을프로테오믹스기법을이용하여확인하고자하였다. 과립막세포단백질의수집도축직후의돼지에서난소를회수하여 37 의생리식염수에침적하여 2 3 시간이내에실험실로운반하였다. 난포액은 10 ml 주사기에 18G 주사침을이용하여직경 2 4 mm 의종속난포와 6 10 mm 의우세난포로부터각각흡입, 회수하였다. 이를 70 μm filter 를이용하여난자를제거하고, 10 분간 800 g 로원심분리하여상층액을제거하였다. 그후 NH 4-Tris buffer[tris(t1503, Sigma), NH 4Cl, DDW, ph 7.6] 10 ml 를넣고피펫팅한후 5 분간 800 g 로원심분리하여상층액을제거하는방법을 3 4 회시행함으로써혈구세포를파괴, 제거시켰다. 그후과립막세포만남은것을 PBS 로 2 3 회세척하여, M-PER Mammalian Protein Extraction Reagent(Thermo scientific, JG123423, USA) 1ml 를첨가하여 시간동안 4 에서단백질을용해하여 12,000 g 에서 5 분간원심분리후상층액을회수하여 -96 에보관하여사용하였다. 이차원전기영동 Clean-up kit(amersham Biosciences, , USA) 를이용해단백질을정제하여 Bradford 법을이용하여총단백질량이 200 μg/μl 가되도록정량하여 Rehydration buffer(ge Healthcare, AE, Sweden) 에녹여총 250 μl 가되도록하였다. 이것을 18 cm 인 NL 3 10 의 IPG strip(amersham Biosciences, , Sweden) 을이용하여 20 에서 16 시간동안흡수시켰다. Isoelectric Focusing(IEF) 는 Protean IEF cell(bio-rad) 을이용하여 250 V 에서 2 시간, 8,000 V 에서 3 시간, 8,000 V 에서 60,000 Vhr 까지채우는방법으로시행하였다. IEF 가끝난 IPG strip 을 equilibration buffer[1.5m Tris(pH 8.8), urea, glycerol, SDS, DDW] 5 ml 에 DTT 0.08 g 을섞은용액에침지하여실온에서 15 분간반응시킨후, equilibration buffer 5 ml 에 iodoacetamide(iaa) 0.1 g 을섞은용액에침지하여실온에서 15 분간반응시켜평형시켰다. SDS-PAGE 는 10% acrylamide gel 에 IPG strip 을 sample 로이용하여 150 V 에서 10 분간, 30mA 에서 10 시간동안전기영동하였다. 전기영동이끝난 gel 은 silver staining kit(ge Healthcare, , Sweden) 을이용하여염색한후, 이미지분석프로그램인 Melanie 7 로 spots 를분석하고, 육안으로검토하였다. 단백질동정 Gel 조각을 destain solution(30 mm potassium ferricyanide, 100 mm sodium thiosulfate, 1:1) 100 μl 에 10 분간침지한후, destain solution 을제거하고 dh 2O 로 15 분간세척하여수용성으로변한은을제거하였다. Gel 을건조시키기위해 100% acetonitrile(acn) 에 5 분간침지한후, ACN 을제거하고 분간 speed-vac 하였다. Gel 에 cold trypsin solution[nh 4bicarbonate(pH 8.8), promega sequencing grade modified trypsin 20 μg/ml] 20 μl 를 30 분간완전히흡수시킨후, 37 에서 시간동안배양하였다. 그후, Gel 조각을 0.1% trifluroacetic acid(tfa) 35 μl 에넣고 60 분간진탕혼합하여상층액을회수하는작업을 2 회반복하였고, 회수된두상층액을혼합하여 speed-vac 으로완전히말려펩타이드를추출하였다. 그후, 0.1% TFA 를첨가하여추출된펩타이드를용해하였다. 0.1% TFA, acetonitrile 혼합액과 0.1% TFA, acetonitrile, 물혼합액으로 Zip-Tip(Zip-Tip C18, millipore) 을세척하였고, 0.1% TFA, 물혼합액으로 Zip-Tip 을평형시킨후, 0.1% TFA 10 μl 에녹인샘플을피펫팅으로빠르게 Zip-Tip 을통과시켜 resin 으로샘플이붙게하였다. 그후 0.1% TFA, 물의혼합액으로 Zip-Tip 을세척하였고, Zip-Tip 으로부터샘플을 alpha-cyano-4-hydroxycinnamic acid(0.3% TFA, 50% acetonitrile) 1.8 μl 에용해하여 MA- LDI-TOF 에이용하였다. MALDI-TOF 는 applied biosystems voyager system 4372 를이용하였으며, 모든결과는 matrix 로 alpha-cyano-4-hydroxycinnamic acid 를이용해 reflector-positive mode 로얻었다. MALDI-TOF 를통해얻어진스펙트럼을이용하여 MOSCOT program 으로 NBCInr 에서 database 검색을실시하였다. 난포의발달단계에따른돼지난포과립막세포의단백질패턴을확인하기위해이차원전기영동을실시한후 Melanie 7 과육안검토를통해이미지분석을하였다 (Fig. 1). 그중종속난포에서보다우세난포에서많이발현된 Spots 7 개를선별하여 MALDI-TOF 를이용해단백질을동정하였다. 그결과, similar to zinc/ling finger protein 3 precursor(ring finger protein 203), angiomotin, heat shock 60kDa protein 1(chaperonin) isoform 1(HSP60), similar to transducin-like enhancer protein 1(TLE1) 및 Fig. 1. Two dimensional gel image of protein from granulosa cell within 2 4 mm follicles(a) and granulosa cell within 6 10 mm follicles(b) in pig ovaries. Proteins were isolated from A and B and 200 g of total protein was loaded to the 2-DE gel electrophoresis. First dimension used 18 m, ph 3 10 NL strip and second dimension used 10% acrylamide gel. Loaded gels were visualized by the silver staining. When size of spots of B as contrasted with A spots were selected up-regulated proteins in B.

3 돼지난포과립막세포내단백질변화 185 Table 1. Up-regulated proteins in granulosa cells within 6 10 mm follicles in pig ovary Spot No. Protein name Accession No. Type of analysis Score % Coverage MW (Da) pi 1 Similar to zinc/ling finger protein 3 precursor (RING finger protein 203) MALDI-TOF/TOF Angiomotin MALDI-TOF/TOF Heat shock 60kDa protein 1(chaperonin) isoform M MALDI-TOF/TOF 2.06e Heat shock 60kDa protein 1(chaperonin) isoform M MALDI-TOF/TOF Heat shock 60kDa protein 1(chaperonin) isoform M MALDI-TOF/TOF 1.19e Similar to transducin-like enhancer protein MALDI-TOF/TOF SH3 and PX domains 2A MALDI-TOF/TOF (A) (B) Fig. 2. MALDI-TOF MS peptide mass spectrem of spot No. 6 from Fig. 1 (A). The protein corresponding to spot No. 6 in Table 1 by searched database (B). The resulting peptides were then extracted and their masses were measured by MALDI-TOF MS.

4 186 채인순등 SH3 와 PX domains 2A(SH3PXD2A) 으로확인되었다 (Table 1). 이가운데 HPS60 은단백질산화에의해 3 개의 spots 으로나뉘어확인되었는데 6 반복에걸쳐모두동일하게나타났다. 또한펩타이드서열을확인하기위해 MALDI- TOF 에서나온스펨트럼을이용해 Mascot 에서 NCBInr 의 database 를검색하였고, score 값을통해 spot No. 6 과가장상응하는단백질이 TLE1 임을확인하였다. 이단백질 spot 은 peptide mass mapping 을확인하였을때전체 peptide 절편의 15.9% 를덮음으로써입증되었다 (Fig. 2). 작용, 분화와같은다양한세포의기능에서중요한역할을수행하는 PX 단백질을붙이는 domains 이다. 따라서, 이상의증가된단백질들의난소내에서기능에대한기존의보고는없었지만연구의결과로보아이단백질들이난포의발육과연관되어있는것으로생각되며, 앞으로의연구에서이단백질들의난소난포내에서의기능을연구해야할것이다. 본연구는돼지난소의난포발달단계에따라과립막세포에서발현되는단백질의변화를확인하기위해수행되었다. 연구에서돼지난소의난포직경은체외성숙및수정에일반적으로이용되는미성숙난자를포함하는 2 4 mm 와난소내에서난포의발달이이루어져배란을준비하는 6 10 mm 로설정하였다. 각각의난포에서채취한과립막세포의단백질을이차원전기영동을통해분리하고 MALDI-TOF 를이용해동정하여 NCBInr 에서 database 를검색한결과, 우세난포에서증가하는경향을보인 7 개 spot 들은 similar to zinc/ling finger protein 3 precursor(ring finger protein 203), angiomotin, heat shock 60kDa protein 1(chaperonin) isoform 1(HSP60), similar to transducin-like enhancer protein 1(TLE1) 및 SH3 와 PX domains 2A(SH3PXD2A) 단백질로확인되었다. 이우세난포에서증가된단백질들은대체적으로세포이동과관련되어있었다. Similar to zinc/ling finger protein 3 precursor(ring finger protein 203) 는일제히 ubiqutination 효소에붙어그들을기질로하여 ligase 로기능하는것으로보고되었다 (Joazeiro 와 Weissman, 2000). Angiomotin 은혈관신생에관련된단백질로세포의이동과밀접한관계가있으며, HSP60 은미토콘드리아단백질수송에관련하여단백질의접힘을촉진하고소수성부분은막을통해서생기는수송에서단백질의접히지않은형태를유지하는원인이된다 (Koll 등, 1992). 또한, DNA 대사에관여하여미토콘드리아 DNA 의전사와복제에연관되어있으며 (Hansen 등, 2003), 세포막에서의세포사멸을예방하는역할을수행하며열충격이나독성, 스트레스환경하에서증가하는경향을보이는것으로보고되었다 (Vargas- Parada 등, 2003; Vittorio Calabrese 등 2007). 한편, TLE1 은 Wnt signal 에관여하며염증유발인자의발현을억제하고 (Fisher 등, 1998; Parkhurst, 1998; Chen 등, 2000; Buscarlet 등, 2007), 태아의발달동안신경조직의생성과관계가있는 Amino-terminal enhancer of split(aes) 에연관되어있으며, 스테로이드호르몬수용체의수많은핵호르몬수용체 family 인 estrogen-related receptor gamma(esrrg) 의보조활성인자로서특별한기능을하는것으로알려져있다. SH3PXD2A 는효소의조절, 신호전달물질의세포내이동, multiprotein complex 생성을조절하는 PxxP motifs 를조절하는 SH3 와세포의성장, 사멸, 세포이동, 대식 본연구를수행하는데있어서단백질분석에협력하여주신강원대학교동물자원공동연구소에감사드립니다. 1. Buscarlet M, Stifani S (2007): The 'Marx' of Grougho on development and disease. Trends Cell Biol 17: Chen G, Courey AJ (2000): Groucho/TLE family proteins and transcriptional repression. Gene 249: Downs S (1995): The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and metiotic control in the isolated mouse oocyte. Dev Biol 167: Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM (1998): The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mole. Endocrinol. 12: Eppig JJ, Wigglesworth K, Pendola F, Hirao Y (1997): Murine oocytes suppress expression of luteinizing hormone receptor messenger rionucleic acid by granulosa cells. Biol Reprod 56: Eppig JJ, Wigglesworth K, Pendola FL (2002): The mammalian oocyte orchestrates the rate of ovarian follicular development. Proceedings of the National Academy of Science. 99(5): Erickson CF, shimasaki S (2001): The physiology of folliculogenesis: the role of novel growth factors. Fertil. Steril. 76(5): Fisher AL, Caudy M (1998): Groucho proteins: transcriptional co-repressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev 12: Hansen TJ, Bross P, Westergaard M, Nielsen MN, Eiberg H, Borglum AD, Mogensen J, Kristiansen K, Bolund L, Niels G (2003): Genomic structure of the human mitochondrial chaperonin genes: HSP60 and HSP10 are localized head to head on chromosome 2 separated by a bidirectional promoter. Human Genetics 112: Hsueh AJW, McGee EA, Hayashi M, Hsu SY (2000): Hormonal regulation of early follicle development

5 돼지난포과립막세포내단백질변화 187 in the rat ovary. Mole Cellular Endocrinol 163: Joazeiro CA, Weissman AM (2000): Ring finger proteins: mediators of ubiquitin ligase activity. Cell 102: Koll H, Guiard B, Rassow J, Ostermann J, Horwich A, Neupert W, Hartl FU (1992): Antifolding activeity of HSP60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell 68: Matzuk MM, Burns KH, Viveiros MM, Eppig JJ (2002): Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296: Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF, Shimasaki S (2000): Bone morphogenetic protein-15. Identification of target cells and biological functions. J Biol Chem 275(50): Parkhurst SM (1998): Groucho: making its Marx as a transcriptional co-repressor. Trends Genet 14: Parrott JA, Skinner MK (1998): Thecal cell granulosa cell interactions involve a positive feedback loop among keratinocyte growth factor, hepatocyte growth factor, and kit ligand during ovarian follicuar development. Endocrinology 139(5): Tisdall DJ, Fidler AE, Smith P, Quirke LD, Stent VC, Heath DA, McNatty KP (1999): Stem cell factor and c-kit gene expression and protein localisasion in the sheep ovary during fetal development. J Reprod Dev 116: Vanderhyden BC (1996): Oocyte-secreted factors granulosa cell steroidogenesis. Zygote. 4: Vargas-Parada L, Solis C (2003): Heat shock and stress response of Teania solium and T. crassiceps. Parasitology 211: Vittorio C, Cesare M, Agrippino R, Marzia P, Chiara C, Carlo DM, Allan B, Anna M, Giuffrida S (2007): In vivo induction of heat shock proteins in the substantia nigra following L-DOPA administration is associated with increased activity of mitochondrial complex I and nitrosative stress in rat : regulation by glutathione redox state. Journal of Neyrochemistry 101(3): Yoshida H, Takakura H, Kunisada T, Okamura H, Nishikawa S (1997): Step-wise requirement of c-kit tyrosine kinase in mouse ovarian follicle development. Dev Biol 184: ( 접수일자 : / 채택일자 : )

(Establishment and Management of Proteomics Core Facility)

(Establishment and Management of Proteomics Core Facility) 1 sample running on SDS-PAGE Gel (10well) 163570 10 16357 1D size marker 225500 100 2255 Buffer(20X, 500ml) 119900 20 5995 Staining